Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Motor-free mitochondrial presequence translocase drives membrane integration of preproteins


The mitochondrial inner membrane is the central energy-converting membrane of eukaryotic cells. The electrochemical proton gradient generated by the respiratory chain drives the ATP synthase. To maintain this proton-motive force, the inner membrane forms a tight barrier and strictly controls the translocation of ions1. However, the major preprotein transport machinery of the inner membrane, termed the presequence translocase, translocates polypeptide chains into or across the membrane2,3,4,5,6,7,8,9. Different views exist of the molecular mechanism of the translocase, in particular of the coupling with the import motor of the matrix8,10,11. We have reconstituted preprotein transport into the mitochondrial inner membrane by incorporating the purified presequence translocase into cardiolipin-containing liposomes. We show that the motor-free form of the presequence translocase integrates preproteins into the membrane. The reconstituted presequence translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. Thus, the minimal system for preprotein integration into the mitochondrial inner membrane is the presequence translocase, a cardiolipin-rich membrane and a membrane potential.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The motor form of the mitochondrial presequence translocase (TIM23CORE) is disrupted in the tim23-76 mutant, whereas the sorting form (TIM23SORT) remains intact.
Figure 2: Reconstitution of the TIM23SORT complex into proteoliposomes.
Figure 3: Reconstituted TIM23SORT forms a presequence-sensitive channel.
Figure 4: Protein insertion by the reconstituted TIM23SORT complex.
Figure 5: Membrane insertion of cytochrome c1 precursor does not depend on the presence of respiratory chain complexes.

Similar content being viewed by others


  1. Palmieri, F. et al. Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins. Biochim. Biophys. Acta 1757, 1249–1262 (2006).

    Article  CAS  Google Scholar 

  2. Dolezal, P., Likic, V., Tachezy, J. & Lithgow, T. Evolution of the molecular machines for protein import into mitochondria. Science 313, 314–318 (2006).

    Article  CAS  Google Scholar 

  3. Jensen, R. E. & Johnson, A. E. Opening the door to mitochondrial protein import. Nature Struct. Biol. 8, 1008–1010 (2001).

    Article  CAS  Google Scholar 

  4. Oka, T. & Mihara, K. A railroad switch in mitochondrial protein import. Mol. Cell 18, 145–146 (2005).

    Article  CAS  Google Scholar 

  5. Koehler, C. M. New developments in mitochondrial assembly. Annu. Rev. Cell Dev. Biol. 20, 309–335 (2004).

    Article  CAS  Google Scholar 

  6. Schnell, D. J. & Hebert, D. N. Proteins translocons: multifunctional mediators of protein translocation across membranes. Cell 112, 491–505 (2003).

    Article  CAS  Google Scholar 

  7. Wickner, W. & Schekman, R. Protein translocation across biological membranes. Science 310, 1452–1456 (2005).

    Article  CAS  Google Scholar 

  8. Neupert, W. & Herrmann, J. M. Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 (2007).

    Article  CAS  Google Scholar 

  9. Rehling, P., Brandner, K. & Pfanner, N. Mitochondrial import and the twin-pore translocase. Nature Rev. Mol. Cell. Biol. 5, 519–530 (2004).

    Article  CAS  Google Scholar 

  10. Chacinska, A. et al. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell 120, 817–829 (2005).

    Article  CAS  Google Scholar 

  11. Tamura, Y. et al. Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria. J. Cell Biol. 174, 631–637 (2006).

    Article  CAS  Google Scholar 

  12. Mokranjac, D., Popov-Celeketic, D., Hell, K. & Neupert, W. Role of Tim21 in mitochondrial translocation contact sites. J. Biol. Chem. 280, 23437–23440 (2005).

    Article  CAS  Google Scholar 

  13. van der Laan, M. et al. A role for Tim21 in membrane-potential-dependent preprotein sorting in mitochondria. Curr. Biol. 16, 2271–2276 (2006).

    Article  CAS  Google Scholar 

  14. Matouschek, A., Pfanner, N. & Voos, W. Protein unfolding by mitochondria: the Hsp70 import motor. EMBO Rep. 1, 404–410 (2000).

    Article  CAS  Google Scholar 

  15. Liu, Q., D'Silva, P., Walter, W., Marszalek, J. & Craig, E. A. Regulated cycling of mitochondrial Hsp70 at the protein import channel. Science 300, 139–141 (2003).

    Article  CAS  Google Scholar 

  16. Chacinska, A. et al. Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM–TIM–preprotein supercomplex. EMBO J. 22, 5370–5381 (2003).

    Article  CAS  Google Scholar 

  17. Truscott, K. N. et al. A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nature Struct. Biol. 8, 1074–1082 (2001).

    Article  CAS  Google Scholar 

  18. Kozany, C., Mokranjac, D., Sichting, M., Neupert, W. & Hell, K. The J-domain related co-chaperone Tim16 is a constituent of the mitochondrial TIM23 preprotein translocase. Nature Struct. Mol. Biol. 11, 234–241 (2004).

    Article  CAS  Google Scholar 

  19. Dekker, P. J. et al. The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44. EMBO J. 16, 5408–5419 (1997).

    Article  CAS  Google Scholar 

  20. Rapaport, D. et al. Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria. Mol. Biol. Cell 12, 1189–1198 (2001).

    Article  CAS  Google Scholar 

  21. Model, K. et al. Multistep assembly of the protein import channel of the mitochondrial outer membrane. Nature Struct. Biol. 8, 361–370 (2001).

    Article  CAS  Google Scholar 

  22. Görlich, D. & Rapoport, T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615–630 (1993).

    Article  Google Scholar 

  23. Meinecke, M. et al. Tim50 maintains the permeability barrier of the mitochondrial inner membrane. Science 312, 1523–1526 (2006).

    Article  CAS  Google Scholar 

  24. Glick, B. S. et al. Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism. Cell 69, 809–822 (1992).

    Article  CAS  Google Scholar 

  25. Zwizinski, C. & Neupert, W. Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences. J. Biol. Chem. 258, 13340–13346 (1983).

    CAS  PubMed  Google Scholar 

  26. Pfanner, N., Hartl, F. U., Guiard, B. & Neupert W. Mitochondrial precursor proteins are imported through a hydrophilic membrane environment. Eur. J. Biochem. 169, 289–293 (1987).

    Article  CAS  Google Scholar 

  27. Schatz, G. The magic garden. Annu. Rev. Biochem. 76, 673–678 (2007).

    Article  CAS  Google Scholar 

  28. Kozjak, V. et al. An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane. J. Biol. Chem. 278, 48520–48523 (2003).

    Article  CAS  Google Scholar 

  29. Ryan, M. T., Voos, W. & Pfanner, N. Assaying protein import into mitochondria. Methods Cell Biol. 65, 189–215 (2001).

    Article  CAS  Google Scholar 

  30. Hartl, F. U., Ostermann, J., Guiard, B. & Neupert, W. Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell 51, 1027–1037 (1987).

    Article  CAS  Google Scholar 

Download references


We are grateful to A. Chacinska and C. Meisinger for helpful discussion. This work was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereiche 388, 431 and 746, an EMBO long-term fellowship (M.v.d.L.), Gottfried Wilhelm Leibniz Program, Max Planck Research Award, Alexander von Humboldt Foundation, Bundesministerium für Bildung und Forschung and the Fonds der Chemischen Industrie.

Author information

Authors and Affiliations



M.v.d.L. established the reconstituted import system, performed and analysed import experiments. M.M. performed and analysed reconstitution/electrophysiological studies. J.D. and M.v.d.L. characterized the mutant TIM23 allele. D.P.H. performed and analysed coimmunoprecipitation experiments. M.L. performed and analysed experiments relating to Tim23 assembly in tim21 mutant mitochondria. I.P. purified the presequence translocase complex. B.G. generated conditional yeast mutant strains. R.W. supervised electrophysiological analysis and participated in data analysis. N.P. and P.R. led the project and participated in data analysis. All authors participated in the preparation of the manuscipt.

Corresponding authors

Correspondence to Richard Wagner or Nikolaus Pfanner.

Supplementary information

Supplementary Title

Supplementary Figures S1, S2, S3 and Methods (PDF 528 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

van der Laan, M., Meinecke, M., Dudek, J. et al. Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nat Cell Biol 9, 1152–1159 (2007).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing